Comprehensive Assessment of Eleven de novo HiFi Assemblers on Complex Eukaryotic Genomes and Metagenomes

Background: Pacific Bioscience HiFi sequencing technology generates long reads (>10 kbp) with very high accuracy (less than 0.01% sequencing error). While several de novo assembly tools are available for HiFi reads, there are no comprehensive studies on the evaluation of these assemblers. Results: We evaluated the performance of eleven de novo HiFi assemblers on (i) real data for three eukaryotic genomes, (ii) 34 synthetic datasets with different ploidy, sequencing coverage levels, heterozygosity rates and sequencing error rates, (iii) one real metagenomic dataset, and (iv) five synthetic metagenomic datasets with different composition abundance and heterozygosity rates. The nine assemblers were evaluated using QUAST (Quality Assessment Tool) and BUSCO (Benchmarking Universal Single-Copy Ortholog). We also used several additional criteria, namely, completion rate, single-copy completion rate, duplicated completion rate, average proportion of largest category, average distance difference, quality value, run-time and memory utilization. On complex eukaryotic genomes, Hifiasm had a clear advantage over the other assemblers in all tested experiments. On synthetic datasets, Hifiasm, HiCanu, and HiFlye performed equally well. Shasta and Peregrine had good performance across varying ploidy, but required high computational resources. On metagenomic datasets, Hifiasm-meta demonstrated a clear advantage over other assemblers. Conclusion: We carried out a comprehensive benchmarking study of commonly used assemblers on complex eukaryotic genomes and metagenomes. Our study will help the research community to choose the most appropriate assembler for their data and identify possible improvements in assembly algorithms. ### Competing Interest Statement The authors have declared no competing interest.